APPLE-MS: A affinity purification-mass spectrometry method assisted by PafA-mediated proximity labeling

While affinity purification-mass spectrometry (AP-MS) has significantly advanced protein-protein interaction (PPI) studies, its limitations in detecting weak, transient, and membrane-associated interactions remain. To address these challenges, we introduced an innovative proteomic method termed Affinity Purification coupled Proximity LabEling-Mass Spectrometry (APPLE-MS), which combines the high specificity of Twin-Strep-tag enrichment with PafA-mediated proximity labeling. This method achieves unprecedented sensitivity while maintaining high specificity (4.07-fold over AP-MS). APPLE-MS also revealed the dynamic mitochondrial interactome of SARS-CoV-2 ORF9B during antiviral responses, while endogenous PIN1 profiling uncovered novel roles in DNA replication. Notably, APPLE-MS enabled in situ mapping of GLP-1 receptor complexes, demonstrating its unique capabilities for membrane PPI studies. This versatile method advances interactome research by providing comprehensive, physiologically relevant PPI networks, opening new opportunities for mechanistic discovery and therapeutic targeting. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/652320v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@aaf55borg.highwire.dtl.DTLVardef@1d25af1org.highwire.dtl.DTLVardef@150c1acorg.highwire.dtl.DTLVardef@db0d1c_HPS_FORMAT_FIGEXP M_FIG C_FIG MOTIVATION - Cell Reports Methods onlyAffinity purification-mass spectrometry (AP-MS) has become a widely used method for capturing high-affinity protein-protein interactions (PPIs). However, it still faces several limitations, including high levels of non-specific binding, challenges in detecting weak interactions, and limited ability to identify PPIs at the cell surface in situ. To overcome these challenges, we introduce a modular AP-MS method enhanced by PafA-mediated proximity labeling, which improves both the specificity and sensitivity of PPI detection in a single, streamlined workflow. SIGNIFICANCE - Cell Chemical Biology onlyProximity labeling is a powerful tool in chemical biology, providing critical support for studying protein function, cell signaling, and disease mechanisms by chemically labeling and capturing biomolecular interactions. In this study, we introduce a modular affinity purification-mass spectrometry (AP-MS) method that integrates proximity labeling. This method leverages the proximity-dependent enzymatic activity of PafA to covalently attach PupE to nearby proteins, in combination with the high-affinity interaction between Twin-Strep-tag and streptavidin to achieve efficient binding. This innovative approach enables precise identification of protein-protein interactions (PPIs), offering a powerful tool for capturing weak and transient interactions that are often missed by traditional AP-MS techniques. By enhancing both the specificity and efficiency of protein labeling, our work provides a robust chemical biology strategy to elucidate complex protein interactions, which are essential for understanding cellular processes and developing targeted therapeutic strategies, offering new insights into protein function and interaction dynamics in both health and disease.